Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein-disulfide isomerase with superoxide dismutase 1

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.     

Brain β-Amyloid Accumulation in Transgenic Mice Expressing Mutant Superoxide Dismutase 1

Oxidative stress is implicated in both the deposition and pathogenesis of -amyloid (A) protein in Alzheimers disease (AD). Accordingly, overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) in neuronal cells and transgenic AD mice reduces A toxicity and accumulation. In contrast, mutations in SOD1 associated with amyotrophic lateral sclerosis (ALS) confer enhanced pro-oxidative enzyme activities. We therefore examined whether ALS-linked mutant SOD1 overexpression in motor neuronal cells or transgenic ALS mice modulates A toxicity or its accumulation in the brain. Aggregated, but not freshly solubilised, substrate-bound A peptides induced degenerative morphology and cytotoxicity in motor neuron-like NSC-34 cells. Transfection of NSC-34 cells with human wild-type SOD1 attenuated A-induced toxicity, however this neuroprotective effect was also observed for ALS-linked mutant SOD1. Analysis of the cerebral cortex, brainstem, cerebellum and olfactory bulb from transgenic SOD1^G93A mice using enzyme-linked immunosorbent assay of acid–guanidine extracts revealed age-dependent elevations in A levels, although not significantly different from wild-type mouse brain. In addition, brain amyloid protein precursor (APP) levels remained unaltered as a consequence of mutant SOD1 expression. We therefore conclude that mutant SOD1 overexpression promotes neither A toxicity nor brain accumulation in these ALS models.     

Generation of B. nigra-B. rapa chromosome addition stocks: cytology and microsatellite markers (SSRs) based characterization

To improve Brassica nigra, the B-genome donor for Brassica juncea through selective introgression of useful variation from A-genome chromosomes, B. nigra-B. rapa chromosome addition stocks were successfully synthesized for the first time. Resynthesized B. juncea was used as B-genome donor species and A-genome addition stocks were developed by hybridizing sesquidiploid plant (ABB) as female and using B. nigra as the male parent. Various cycles of backcrossing and/or selfing were utilized to isolate plants carrying addition of three A-genome chromosomes in the background of B. nigra. These chromosome addition stocks were characterized by chromosome counts, pollen and seed fertility and chromosome specific microsatellite (SSRs) markers. The chromosome number in different backcross/self generations ranged between 2n=26 and 2n=19 with relatively high frequency of univalents (8-10I) at in meiotic configurations observed, suggesting the role of preferential transmission of A-genome chromosomes. SSRs analysis revealed that B. rapa chromosomes 3 and 4 were the first to get eliminated followed by chromosome 10. Remaining chromosomes were maintained till BC(1)F(4). However, second cycle of backcrossing (BC(2)) led to the elimination of chromosome numbers 1 and 2. BC(2)F(2) plants carried the chromosome numbers 6, 7, 8 and 9. Generation BC(3) having plants with 2n=19 carried chromosome numbers 6, 7 and 8. It is possible that chromosomes 6, 7 and 8 had higher transmission frequency and these were better tolerated by the B. nigra genome.     

Design and application of a peptide nucleic acid sequence targeting the p75 neurotrophin receptor

Novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential therapeutic strategy for amyotrophic lateral sclerosis (ALS) were designed, synthesised and evaluated against phosphorothioate oligonucleotide sequences (PS-ODN). An 11-mer antisense PNA directed at the initiation codon dose-dependently inhibited p75NTR expression and death signalling by nerve growth factor in Schwann cell cultures. Inhibition of p75NTR production was not detected in cultures treated with the nonsense PNA or antisense PNA directed at the 3'-terminus sequence. The 19-mer PS-ODN sequences also failed to confer any activity against p75NTR but, unlike the PNA sequences, were toxic in vitro at comparable doses.     

Effective Use of Marine Algal Products in the Management of Plant-Parasitic Nematodes

Algal extracts were ineffective against Meloidogyne spp., Panagrellus redivivus, and Neoaplectana carpocapsae at 1.0% aqueous concentrations, with the exception of Spatoglossum schroederi. S. schroederi killed Meloidogyne incognita, M. javanica, M. acrita, and Hoplolaimus galeatus at concentrations of 1.0, 0.75, and 0.50%. Extracts from S. schroederi at a concentration of 1.0% were ineffective against Hirschmanniella caudacrena and Belonolaimus longicaudatus. Spatoglossum schroederi, Botryocladia occidentalis, and Bryothamnion triquestrum when used as soil amendments at 0.5-1.0% concentrations (by weight) produced significant reduction of root gall development in tomato plants infected with M. incognita. Tomato plant growth was significantly improved by these algae, as well as by Caulerpa prolifera. Soil amendments of S. schroederi at concentrations of 0.5 and 1.0% significantly reduced root galling of tomato infected with M. incognita, M. arenaria, and M. javanica. Tomatoes grown in algal-soil mixture produced significantly heavier shoots and roots than plants raised in autoclaved soil. No significant differences in root-knot indices, nor in fresh and dry weights of tomato, were noted between the two concentrations of algal-soil mixture.     

Antisense peptide nucleic acid-mediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice

Re-expression of the death-signalling p75 neurotrophin receptor (p75NTR) is associated with injury and neurodegeneration in the adult nervous system. The induction of p75NTR expression in mature degenerating spinal motor neurons of humans and transgenic mice with amyotrophic lateral sclerosis (ALS) suggests a role of p75NTR in the progression of motor neuron disease (MND). In this study, we designed, synthesized and evaluated novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential gene knockdown therapeutic strategy for ALS. An 11-mer antisense PNA directed at the initiation codon, but not downstream gene sequences, dose-dependently inhibited p75NTR expression and death-signalling by nerve growth factor (NGF) in Schwann cell cultures. Antisense phosphorothioate oligonucleotide (PS-ODN) sequences used for comparison failed to confer such inhibitory activity. Systemic intraperitoneal administration of this antisense PNA to mutant superoxide dismutase 1 (SOD1G93A) transgenic mice significantly delayed locomotor impairment and mortality compared with mice injected with nonsense or scrambled PNA sequences. Reductions in p75NTR expression and subsequent caspase-3 activation in spinal cords were consistent with increased survival in antisense PNA-treated mice. The uptake of fluorescent-labelled antisense PNA in the nervous system of transgenic mice was also confirmed. This study suggests that p75NTR may be a promising antisense target in the treatment of ALS.     

Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts

High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.     

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25–30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.